WO2019067464A1 - Agents thérapeutiques spécifiquement administrés par des exosomes pour le traitement du cancer - Google Patents

Agents thérapeutiques spécifiquement administrés par des exosomes pour le traitement du cancer Download PDF

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WO2019067464A1
WO2019067464A1 PCT/US2018/052704 US2018052704W WO2019067464A1 WO 2019067464 A1 WO2019067464 A1 WO 2019067464A1 US 2018052704 W US2018052704 W US 2018052704W WO 2019067464 A1 WO2019067464 A1 WO 2019067464A1
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cells
mrna
evs
exosomes
prodrug
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Jing-Hung Wang
Alexis FORTERRE
A. C. Matin
Alain Delcayre
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to US16/649,083 priority Critical patent/US11517530B2/en
Publication of WO2019067464A1 publication Critical patent/WO2019067464A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/05—Oxidoreductases acting on NADH or NADPH (1.6) with a quinone or similar compound as acceptor (1.6.5)
    • C12Y106/05005—NADPH:quinone reductase (1.6.5.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the active agent in the exosome is a nucleic acid, such as an interfering RNA or an mRNA.
  • the active agent loaded into the exosome is selected from a DNA, an RNA, an mRNA, an siRNA or miRNA, a polypeptide/protein, an antibiotic, a prodrug and a small molecule compound.
  • the active agent is an mRNA.
  • the mRNA encodes an enzyme that converts a prodrug to a drug.
  • the enzyme is ChrR.
  • the prodrug is selected from a dinitrobenzamide, a nitroaniline- based alkylating agent, and a quinone.
  • the targeting moiety is an antibody or functional fragment thereof (e.g., scFv). In some embodiments, the targeting moiety is an extracellular receptor-targeting scFv antibody. In some embodiments, the active agent is a polypeptide. In some embodiments, the active agent is a small molecule.
  • FIG. 2A shows a schematic representation of the HER2 receptor- targeting ML39 chimeric protein (EVHB).
  • FIG. 2B shows a NanoSight nanoparticle analysis of the EVs, where concentration of particles/ml is plotted vs. size (nm).
  • FIG. 2C shows Western blots of extracted protein from EVs or whole cells of HEK293 cells transfected with pEVC1C2HER plasmid, or the empty plasmid (p6ml_SC1C2; control.
  • FIG. 2D shows the predicted protein structure of EVHB, including the ML39 scFv antibody, the leader sequence, and the C1 and C2 domains.
  • FIG. 3A shows ELISA detection of HER2 receptor binding activity of directed EVs (displaying EVHB) obtained from pEVC1C2HER plasm id-transfected HEK293 cells and of naive EVs obtained from non-transfected HEK293 cells incubated with pure EVHB.
  • FIG. 3B is a schematic representation of EVHB display by EVs from HEK 293 cells (upper left cell containing organelles). The left panel labeled "Transfection” shows cells transfected with pEVC1C2HER plasmid, and EVs obtained from them, with an enlarged EV showing the membrane bilayer.
  • composition and methods employ a new chimeric protein construct, termed EVHB, consisting of: i) leader sequence (LS) for EVHB migration to the exosome surface; ii) high affinity anti-HER2 scFv antibody to target the HER2 receptor; iii) lactadherin C1 -C2 domains, which bind to exosomes by interacting with their surface phosphatidylserine; and iv) His-tag, for EVHB purification.
  • leader sequence LS
  • LS leader sequence
  • scFv antibody to target the HER2 receptor
  • lactadherin C1 -C2 domains which bind to exosomes by interacting with their surface phosphatidylserine
  • His-tag for EVHB purification.
  • HEK293 cells transfected with the plasmid encoding this protein generate exosomes that express EVHB on their surface and have the capability of specifically targeting HER2-positive receptor, cells, and tumors (termed, "targeted” exosomes).
  • exosomes have also been loaded with exogenous mRNA, using the 'zipcode' technology that promotes mRNA entry into exosomes and a commercially available plasmid.
  • the directed and loaded exosomes (called the "EXO-DEPT" exosomes) specifically deliver the mRNA to HER2-positive cells and tumors in mice.
  • the mRNA encodes humanized and improved form of a bacterial enzyme, called HChrR6.
  • the treatment / effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • "Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering the methods of the present disclosure.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds / therapeutic agents of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such carriers can be sterile liquids, such as saline solutions in water, or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • a saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium s tea rate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the carrier if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin.
  • suitable pharmaceutical carriers are a variety of cationic polyamines and lipids, including, but not limited to N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA) and diolesylphosphotidylethanolamine (DOPE).
  • DOTMA N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • DOPE diolesylphosphotidylethanolamine
  • Liposomes are suitable carriers for gene therapy uses of the present disclosure.
  • Such pharmaceutical compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. These terms also include proteins that are post-translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
  • a target cell is a cell expressing an extracellular receptor on its surface. Tumor cells often overexpress such extracellular receptors.
  • a target cell is a mammalian cell, preferably a human cell.
  • a "targeting moiety" having a high affinity for a receptor overexpressed in a disease state can be a binding protein (having a region, such as a complementarity determining region (CDR) that binds to a particular target receptor protein/polypeptide, or a fragment or epitope of the receptor), an antibody, or an antibody fragment (e.g., an scFv antibody).
  • CDR complementarity determining region
  • a targeting moiety is said to specifically bind or selectively bind to the target when the dissociation constant (Kc) is ⁇ 10 "8 M .
  • the targeting moiety may specifically bind the target with high affinity when the KD is ⁇ 10 9 M or KD is ⁇ 10 "10 M.
  • the targeting moiety e.g.,scFv or antibody fragment
  • the targeting moiety may bind to the target receptor or epitope with a KD of between about 10 7 M and about 10 "12 M.
  • the targeting moiety may bind with a KD of 1-2 x 10 ⁇ .
  • antibody and "immunoglobulin” or “Ig” are used interchangeably herein, and is used in the broadest sense and specifically covers, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), anti-receptor antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain anti-receptor antibodies, and fragments of anti-receptor antibodies, as described below.
  • an antibody can be human, humanized, chimeric and/or affinity matured as well as an antibody from other species, for example mouse, rabbit etc.
  • the term "antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed.
  • the specific molecular antigen can be bound by an antibody provided herein includes a receptor polypeptide, receptor fragment or receptor epitope.
  • Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments such as receptor binding fragments) of any of the above, which refers a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • functional fragments e.g., antigen-binding fragments such as receptor binding fragments
  • functional fragments include single-chain Fvs (scFv) (e.g...
  • Non-limiting examples of nucleic acids and polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, primers, single-, double-, or multi-stranded DNA or RNA, genomic DNA, DNA- RNA hybrids, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, oligonucleotides containing modified or non-natural nucleotide bases (e.g., locked- nucleic acids (LNA) oligonucleotides), and interfering RNAs.
  • mRNA messenger RNA
  • cDNA recombinant polynucleotides
  • vectors probes, primers
  • single-, double-, or multi-stranded DNA or RNA genomic DNA
  • DNA- RNA hybrids chemically or biochemically modified, non-natural, or derivatized nucleotide bases
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi(dot)nlm(dot)nih(dot)gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.
  • FASTA Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
  • double stranded RNA refers to nucleic acid molecules capable of being processed to produce a smaller nucleic acid, e.g., a short interfering RNA (siRNA), capable of inhibiting or down regulating gene expression, for example by mediating R A interference "RNAi” or gene silencing in a sequence-specific manner.
  • siRNA short interfering RNA
  • siRNA When a particular siRNA is described herein, it will be clear to the ordinary skilled artisan as to where and when a different but equivalent ⁇ effective interfering nucleic acid may be substituted, e.g., the substation of a short interfering oligonucleotide for a described shRNA and the like.
  • "Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides of a polynucleotide (e.g., an antisense polynucleotide) and its corresponding target polynucleotide.
  • a nucleotide at a particular position of a polynucleotide is capable of hydrogen bonding with a nucleotide at a particular position of a target nucleic acid
  • the position of hydrogen bonding between the polynucleotide and the target polynucleotide is considered to be a complementary position.
  • the polynucleotide and the target polynucleotide are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides that can hydrogen bond with each other.
  • polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure).
  • a polynucleotide can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • an antisense polynucleotide which is 18 nucleotides in length having four noncomplementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid.
  • cytotoxicity is a term well understood in the art and refers to a state in which one or more of a cell's usual biochemical or biological functions are perturbed. These activities include, but are not limited to, metabolism, cellular replication, DNA replication, transcription, translation, and uptake of molecules. "Cytotoxicity” includes cell death and/or cytolysis. Assays are known in the art which indicate cytotoxicity, such as dye exclusion, 3H- thymidine uptake, and plaque assays.
  • Such drugs include, without limitation, 2,5-diaziridinyl-3-(hydroxymethyl)- 6-methyl-1 ,4- benzoquinone; 5-aziridinyl-2,4-dinitrobenzamide (CB 1954); 1 ,4-bis[[2-(dimethylamino) ethyl]amino]-5,8-dihydroxyanthracene-9, 10-dione (AQ4), the dinitrobenzamide mustard compound SN 23862 and related amide-substituted mustard SN 27217; nitroaniline- based alkylating agents as described in U.S.
  • Viruses have commonly been used for gene delivery in GDEPTS, but raise concerns of immune recognition, insertional mutagenesis, and inflammatory toxicity.
  • Extracellular vesicles (EVs, also called exosomes) were used instead. These are small; consist of lipid bilayers; are constitutively generated by most body cells; are largely nontoxic; and can deliver their cargo directly into the cytoplasm, avoiding the endosomal pathway and lysosomal degradation. Their small size mitigates uptake by the reticuloendothelial system, and permits extravasation through vessel fenestrations present in tumors.
  • they may be minimally immunogenic, especially when derived from mesenchymal stem cells or from patient's own, e.g., dendritic cells.
  • HChrR6 As the mRNA is delivered specifically to the tumors, HChrR6 is generated specifically inside them. Consequently, the toxic drug, MCHB, is confined largely to the tumors attaining a high concentration inside them. Advantages of these innovations include the effective killing of cancer cells, avoidance of drug resistance, and prevention of damage to normal tissues. HChrR6 can also activate the prodrug CB1954, currently in clinical trials. Additionally, combined therapy with the two prodrugs can enhance the effectiveness of the treatment.
  • mRNA can be delivered by a plasmid encoding the desired mRNA that is transcribed in the cell, or by delivery of mRNA itself.
  • mRNA encoding the prodrug activating enzyme can be generated by in vitro transcription, and used to load the EV directly with mRNA.
  • In vitro transcription is a simple procedure that allows for template- directed synthesis of RNA molecules, based on the engineering of a template that includes a promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase.
  • composition and methods described herein are generic and the anti-HER2 scFv in the EVHB construct can be easily replaced by another scFv (or other moieties) capable of targeting a different receptor; and the exosomes / EVs can be loaded with another mRNA, biomolecule and/or drug.
  • the approach can be used for therapy of any disease in which a receptor is overexpressed.
  • examples of other receptors overexpressed in cancers are PSMA, bombasin, folate, transferrin, and sigma; many other diseases also overexpress specific receptors.
  • the disclosure also presents methods of screening antibody repertoires comprising 1) providing vesicles bearing at least one target antigen and one marker and 2) isolating antibody-producing cells or particles with defined antigen specificity using the said antigen- and marker-bearing vesicles.
  • Antibodies with defined antigen specificity can then be prepared from isolated antibody-producing cells using known methods of the art.
  • US Patent Application publication 2009/0148460 entitled “Exosome Ligands, their Preparation and Uses” (with inventors Alain DELCAYRE and Jean-Bernard LE PECQ) discloses exosome-specific ligands and compositions comprising the same.
  • the disclosure also relates to methods of generating said ligands and compositions, to methods of using said ligands or compositions, e.g., to block the exosome pathway or to detect and/or characterize exosomes in a sample or subject, as well as to the antigens contacted by said ligands or compositions.
  • the application can be used in experimental, research, therapeutic, prophylactic or diagnostic areas.
  • US Patent Application publication 2017/0051282 entitled “Extracellular Vesicle Methods and Compositions” (with inventors Thomas R. GINGERAS, Sudipto K. CHAKRABORTTY, Ashwin PRAKASH and Gal NECHOOSHTAN, all from Cold Spring Harbor, NY) discloses compositions and methods of producing a therapeutic extracellular cancer vesicle (ECV) comprising an antisense masking oligonucleotide (AMO) having anti-tumor activity and specifically binds to a RNA fragment of a primary RNA transcript of the ECV, wherein the RNA fragment mediates tumor progression, comprising: (a) providing a cancer cell that can produce ECVs; (b) allowing the cancer cell to produce the ECVs; (c) transfecting an AMO in the ECVs; and (d) isolating exosomes produced by the cell, wherein the ECVs comprise the AMO bound to the RNA fragment of a primary RNA transcript.
  • ECV extracellular cancer ve
  • US Patent 8,686, 115 entitled “Compositions and Methods for Quantitatively Monitoring Lipids” (having the sole inventor Wonhwa Cho and assigned to The Board of Trustees of the University of Illinois) is directed to fluorescent lipid binding proteins (FLBPs) comprising a lipid binding domain linked to a fluorophore, whereby the fluorophore's fluorescence emission undergoes a spectral change upon lipid binding, the fluorophore is selected from the group consisting of 2-dimethylamino-6-acyl-naphthalene (DAN) and RED fluorophore, and the lipid binding protein is selected from the group consisting of ENTH domain of epsin 1 , C2 domain of bovine lactadherin, C 1 B domain of protein kinase C-gamma, C2 domain of cytosolic phospholipase A2-beta, and PH domain of Bruton's tyrosine kina
  • a phospholipid such as phosphatidylserine
  • a binding agent including lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of a lactadherin, used to detect the presence of any phospholipid.
  • the prodrug produces a detectable product upon reduction, e.g. a fluorescent compound.
  • the production of the drug from the prodrug can thus be imaged in vitro or in vivo.
  • the tracking of the delivery vehicle and the prodrug provides for methods of in vivo analysis of cancer therapy.
  • Homologs of ChrR are known in the art, e.g. from such bacterium as Shigella boydii; Salmonella enterica; Shigella flexneri; Salmonella typhimurium; Pseudomonas aeruginosa; Streptomyces coelicolor; Bacillus subtilis; Lactococcus lactis, etc.
  • Such homologs usually have at least about 35% amino acid identity with SEQ ID NO: 1 , more usually at least about 45% sequence identity; and may be at least about 80% sequence identity; at least about 85%, at least about 90%, or more.
  • a fragment of a ChrR peptide may be utilized.
  • Peptides of interest include fragments of at least about 50 contiguous amino acids, more usually at least about 100 contiguous amino acids, and may comprise 150 or more amino acids, up to the full length polypeptide. Fragments also included truncated forms of the polypeptide, where deletions may be from about 1 to about 5, to about 10, to about 15, to about 20, to about 25 amino acids, and may extend from residue 1 through 25 at either terminus of the polypeptide, comprising deletions of any length within the region; or may be at an internal location.
  • the sequence of the ChrR polypeptide may be altered in various ways known in the art to generate changes in sequence.
  • the polypeptide will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. Where changes are introduced by shuffling or any other means of random mutation method, the amino acid differences may be greater.
  • the sequence changes may be substitutions, insertions or deletions. Scanning mutations that systematically introduce alanine, or other residues, may be used to determine key amino acids.
  • the polypeptide comprises an amino acid substitution at the position corresponding to amino acid 128 of SEQ ID NO:1. It will be understood by one of skill in the art that the corresponding amino acid can be determined in homologous polypeptides by alignment of the two sequences using conventional algorithms, e.g. BLASTN, CLUSTALW, and the like.
  • the polypeptide comprising an amino acid substitution at the position corresponding to amino acid 128 of SEQ ID NO: 1 further comprises an amino acid substitution at the position corresponding to amino acid 150 of SEQ ID NO: 1 , where the substituted amino acid is other than glycine.
  • substitutions of interest include serine and amino acids that are conservative with respect to serine, including threonine, cysteine, and the like.
  • the polypeptide comprising an amino acid substitution at the position corresponding to amino acid 128 of SEQ ID NO: 1 may further comprise an amino acid substitution at the position corresponding to amino acid 154 of SEQ ID NO: 1 , where the substituted amino acid is other than asparagine.
  • substitutions of interest include threonine and amino acids that are conservative with respect to threonine, including serine, cysteine, and the like.
  • Nitroreductase enzymes can be screened indirectly for Cr(VI) reduction.
  • the colorimetric diphenyl carbazide assay of Greenberg et al Greenberg, A. E., J. J. Connors, D. Jenkins, and M. A. Franson (ed.). 1981. Standard methods for the examination of water and wastewater, 15th ed., p.187-190. American Public Health Association, Washington, D.C.
  • the end product of chromate reduction can be determined using the X-ray absorption near-edge structure (XANES) spectrum.
  • Cr(VI) and Cr(lll) can be distinguished by the pronounced pre edge feature of the former.
  • the fraction of Cr(VI) was calculated by dividing the height of the Cr(VI) pre edge peak by the total absorption; that of Cr(lll) was calculated from the difference between the amount of chromium represented by the pre edge peak and the total absorption jump.
  • an assay is provided wherein the prodrug CNOB is administered to a patient or animal.
  • the fluorescent product is used to monitor the efficacy of activation in an in vivo environment.
  • a laboratory animal is used in such a method, e.g. mouse, rat, rabbit, etc.
  • a human patient is treated in such a method.
  • ChrR polypeptides may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the present disclosure provides compositions and methods of making exosomes directed to HER2-positive cancer. As stated, the disclosure permits using the same protocols for making exosomes directed to other indications in which a receptor is overexpressed and that can benefit by specific delivery of a biomolecule(s)/drug(s). [0143] In some embodiments, the present disclosure provides compositions and methods of making exosomes that can deliver exogenous functional mRNA into recipient cells, and this in a targeted manner. This is the first time that functional mRNA, not indigenous to exosomes, has been successfully delivered to recipient cells; and to a beneficial therapeutic end. As stated, EXO DEPT/prodrug joint treatment arrested the growth of implanted xenografts in mice.
  • HChrR6 mRNA in tumors and other body tissues was quantified by qRT-PCR. MCHB fluorescence was measured and used to quantify the amount of drug delivered. The results indicate that HChrR6 mRNA delivery and MCHB generation capability are restricted to the tumor location.
  • CFSE-labeled EVs were then added to separate groups of wells and incubated in DMEM/EDFBS for 6 hours (37°C) followed by washing in the same medium to remove unbound EVs, and addition of fresh medium (100 ⁇ .
  • Green fluorescence (GFP filter) and phase contrast images were taken (20x magnification; EVOSTM FL Cell Imaging System, Thermo Fisher).
  • SKBR3 cells were seeded in 6-weli plates (1.2x10 6 /well) in DMEM with 10% FBS, and incubated overnight. The medium was then replaced with DMEM/EDFBS containing 1.6x10 9 directed EVs to the wells and incubated (37°C; 4 hours).
  • Cells were washed with ice-cold PBS; 0.2mL/well of cell detaching solution in PBS (Thermo Fisher) was added to dislodge the cells, which were mixed with 1 ml_ DMEM / 10%FBS. Cells were transferred to centrifuge tubes, pelleted by centrifugation (900*g; 4°C; 5minutes), rinsed in 1 ml.
  • RNA extraction from EVs and cells and quantitative RT-PCR RNA was extracted using RNeasy mini kit (QIAGEN, Germany), and quantified by NanoDrop 1000 Spectrophotometer (Thermo Fisher, Wilmington, DE). cDNA was synthesized from RNA of EVs and cells (0.1 and 1 g, respectively), using M-MuLV reverse transcriptase (Taq® RT-PCR kit, New England Biolabs (NEB), Ipswich, MA). To remove RNA, the cDNA was treated with RNase H (NEB).
  • mice Six to seven weeks old female BALB/C athymic nude mice (nu/nu; Charles River Laboratories) were implanted subcutaneously with 0.5mg (60-day release), 17p-estradiol pellets (Innovative Research of America, Sarasota, FL) on the upper dorsal side between the ear and shoulder to support growth of BT474 xenografts, which, in addition to HER2 overexpression, express also the estrogen receptor; a trocar needle was used. On the following day, 10 7 BT474 cells suspended in 100 ⁇ _ of PBS- Matrigel (1 : 1 ; BD Biosciences, San Jose, CA) were subcutaneously injected into mammary fat pad number 9.
  • Tumor size was monitored by caliper at two-day intervals, and tumor volume was calculated using the formula: tumor width 2 x its length/2. After the tumors reached a volume of >150 mm 3 , mice were randomly assigned into groups. EVs were pre-loaded with HChrR6 mRNA. 2x10 9 EVs in 100 L PBS were injected intraperitoneally per mouse per dose. Control mice received an equal amount of PBS.
  • the administration schedule based on previous PK/PD studies of the CNOB/hChrR6 regimen, is provided below.
  • 2B shows a NanoSight analysis of the EVs, where concentration of particles/ml is plotted vs. size (nm).
  • FIG. 1 Schematic representation of EVHB display by EVs from HEK 293 cells (upper left cell containing organelles).
  • the left panel labeled “Transfection” shows cells transfected with pEVC1C2HER plasmid, and EVs obtained from them, with an enlarged EV showing the membrane bilayer.
  • the right panel labeled “Reconstitution” shows non-transfected cells after incubation with pure EVHB which is inserted into the membrane bilayer.
  • 3C Representative fluorescent and phase contrast images of corresponding regions showing the CFSE-labeled directed EV binding to BT474 cells and not to MCF7 cells.
  • 3D Directed EV binding to cells as determined by flow cytometry.
  • Figures 4A - 4G 4A. The design of XPort/HChrR6 plasmid showing key features involved in mRNA packaging into EVs; see text for details.
  • 4B qPCR results showing successful loading of EVs with HChrR6 mRNA. Endogenous EV miR-16 level was determined as control [the Ct value of mRNA corresponds to 2x10 4 copy/EV].
  • 4C In vitro effectiveness of EXO-DEPT EVs.
  • Figure 5 shows the amino acid composition of EVHB, indicating a calculated molecular weight of 68kDa.
  • Immortalized human kidney embryonic (HEK293) cells were transfected with pEVC1C2HER, and the cell-released EVs were isolated; they presented a uniform peak in NanoSight analysis (average size, ca. 30-100 nm; Figure 2B).
  • EVHB was purified both from the EV and cell lysates. Equal protein amounts were analyzed by Western blotting; the expected 68kDa band was seen, which was more intense for the EV fraction (Figure 2C); the band was not found in extracts of non-transfected cells or their EVs.
  • the treatment was started with intraperitoneal injection of 2x10 9 EVs and, 24 hours later, of intravenous injection of CNOB (3.3mg/kg in saline); corresponding controls received PBS (instead of EVs) or saline (instead of CNOB). Further doses (in the same amounts) were administered as shown in Figure 4E.
  • Each mouse was measured at nine time periods, days 0,3, 7, 1 , 6, 9, 24, 28, 32.
  • a linear regression of tumor burden versus day was fit for each mouse, by ordinary least squares, and its slope calculated.
  • Table 2 shows the means, and standard errors (SE) for the slope statistics in the 5 groups, and also the endpoints of two-sided 95% student-t confidence intervals.
  • Group 5 shows strong significance versus 1 ,2,3, and 4; Group 4 shows moderately strong significance versus 1 and 2, and borderline significance versus 3. There are no other significant comparisons.
  • mRNA functionality To test that this mRNA is functional, it was translated in vitro into the HChrR6 protein, again using a standard kit, and the activity of the resulting protein was assayed using a standard procedure (reaction mixture is given in Fig. 6).
  • Fig. 6 shows that HChrR6 synthesized from the IVT mRNA is functional, and it converts CNOB into MCHB.
  • IVT method generates EVs containing more mRNA.
  • HEK293 cells transfected with this IVT HChrR6 mRNA generated EVs containing this mRNA. These are referred to herein as "IVT EVs".
  • the IVT EVs contained much more mRNA than the plasmid EVs (qRT-PCR), such that while with the latter 5,000 EVs are needed to deliver one copy of the mRNA, with the former the same amount can be delivered with as few as 30-40 EVs (Fig. 7).
  • qRT-PCR plasmid EVs
  • Fig. 7 plasmid EVs
  • These EVs were incubated with pure EVHB protein to generate IVT EXO-DEPTs.
  • IVT EXO-DEPTs transfect BT474 cells, causing them to be killed by CNOB. This is shown in Fig. 8, indicating that the IVT mRNA remains functionally competent inside the recipient cells.
  • IVT EXO-DEPTs along with the prodrug CB1954 can suppress tumor growth in mice at a lower EV dose.
  • the prodrug CB1954 (which HChrR6 can activate) is of great interest and is currently in clinical trials.
  • BT474 tumors were orthotopically implanted in nu/nu mice as in Example 1. Both the EVs and CB1954 were injected intraperitoneally; the schedule based on PK considerations and the mRNA content of IVT EXO-DEPTs, as well as early results are shown in Fig. 10. The increased amount of mRNA in these EVs permitted the use of 1.3x10 10 per injection per mouse, much less than the 7.5x10 10 used for plasmid EXO-DEPTs.
  • F ruh be is C
  • Frohlich D Kramer-Albers EM. Emerging roles of exosomes in neuron-glia communication. Front Physiol. 2012; 3: 1 19.
  • cerebrospinal fluid is altered in HER? -positive breast cancer patients with brain metastases and impairment of blood-brain barrier.

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Abstract

L'invention concerne des compositions de vésicule extracellulaire (VE) (également appelées exosomes) pour cibler spécifiquement l'administration d'un agent thérapeutique à des cellules et/ou des tissus particuliers chez un sujet, ainsi que des procédés de fabrication et des procédés d'utilisation desdites compositions. Les compositions et les procédés de l'invention sont utiles pour l'administration ciblée de médicaments dans le traitement de maladies dans lesquelles un récepteur de surface cellulaire est surexprimé, tel que, par exemple, un cancer.
PCT/US2018/052704 2017-09-27 2018-09-25 Agents thérapeutiques spécifiquement administrés par des exosomes pour le traitement du cancer WO2019067464A1 (fr)

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WO2015002956A1 (fr) * 2013-07-01 2015-01-08 Ohio State Innovation Foundation Système de distribution d'exosome
US20150064215A1 (en) * 2013-09-05 2015-03-05 The University Of Hong Kong Therapeutic delivery and expression system, methods and uses thereof
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WO2017201325A1 (fr) * 2016-05-18 2017-11-23 Modernatx, Inc. Combinaisons d'arnm codant pour des polypeptides de modulation immunitaire et leurs utilisations

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US20070254852A1 (en) * 2005-12-20 2007-11-01 Matin A C Improved nitroreductase enzymes
US20160184458A1 (en) * 2013-03-14 2016-06-30 Shire Human Genetic Therapies, Inc. Mrna therapeutic compositions and use to treat diseases and disorders
WO2015002956A1 (fr) * 2013-07-01 2015-01-08 Ohio State Innovation Foundation Système de distribution d'exosome
US20150064215A1 (en) * 2013-09-05 2015-03-05 The University Of Hong Kong Therapeutic delivery and expression system, methods and uses thereof
WO2017201325A1 (fr) * 2016-05-18 2017-11-23 Modernatx, Inc. Combinaisons d'arnm codant pour des polypeptides de modulation immunitaire et leurs utilisations

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